Deubiquitinases are proteases involved in a myriad of cellular pathways in humans. The activity of deubiquitinases is tightly regulated and deregulation leads to human diseases, including cancer, neurodegenerative diseases and autoimmune diseases. The overall objective of the proposed research is to elucidate the molecular mechanisms underlying the regulation and substrate specificity of deubiquitinase A (DUBA), which is one of several deubiquitinases involved in immune responses. Conformational dynamics is implicated in the function of many members of the deubiquitinase family, including DUBA. The activity of DUBA is regulated by phosphorylation in the absence of significant structural changes, which suggests the need to characterize both the structural and dynamical properties of DUBA in order to understand its regulation mechanism. DUBA displays specificities towards more than one ubiquitin chain linkages and the structural basis of substrate recognition is not well defined. Studies on other deubiquitinases suggest the importance of conformational flexibility, which allows substrates of different chain topologies to be accommodated. The proposed research has two specific aims: (1) to characterize the conformational dynamics of DUBA in apo state and to define the functional roles of the observed conformational processes, and (2) to dissect the molecular mechanism of substrate recognition. Solution NMR spectroscopy will be used as the main experimental technique, which provides site-specific information on both structure and dynamics. NMR measurements will be combined with biochemical assays to establish the relationship between the parameters characterizing the conformational dynamics and the catalytic rate constants of wildtype and mutants of DUBA. Structural insights gained from the proposed research can potentially aid in the design of therapeutic strategies for diseases involving deregulation or malfunction of deubiquitinases.